D-Asp upregulates PREP and GluA2/3 expressions and induces p-ERK1/2 and p-Akt in rat testis

in Reproduction
Correspondence should be addressed to S Minucci; Email: sergio.minucci@unicampania.it

*(A Santillo and M Venditti contributed equally to this work)

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D-Aspartate (D-Asp) is an endogenous amino acid that plays a central role in the development of the central nervous system (CNS) and functioning of the neuroendocrine system. In line with its functions, it is abundantly present in the CNS and reproductive systems of vertebrates and invertebrates. It has been implicated in the biosynthesis and/or secretion of hormones and factors that are involved in various reproductive functions, such as GnRH from the hypothalamus and testosterone from the testis. We conducted an in vivo study consisting of acute (i.p. injection of 2 µmol/g body weight) and chronic (15 days drinking solution) administration of D-Asp to adult rats to understand the signaling pathways elicited by D-Asp in the rat testis. We found that D-Asp upregulated the expression of prolyl endopeptidase (PREP), a serine protease having a pivotal role in the regulation of mammalian spermatogenesis and spermiogenesis. Immunofluorescence analysis revealed its overexpression in Leydig cells, Sertoli cells and spermatogonia. Moreover, PREP was found to co-localize with GluA2/3, an AMPA receptor subunit, whose protein expression also increased after D-Asp treatments. Finally, we found a significant increase in ERK and Akt activities in the testis of rats treated with D-Asp. Since PREP is known to be involved in regulating GnRH levels and in germ cell differentiation, we hypothesize D-Asp to play a pivotal role in regulating hormone homeostasis and spermatogenesis through activation of PREP, AMPAR, ERK and Akt.

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  • Fig. 1: Immunofluorescence co-localization analysis for GluA2/3 and PREP in rat testis from controls (a), and at 5 h (b) and 15 days (c) after D-Asp treatment. a, b, c: GluA2/3 (green), PREP (red) and DAPI-fluorescent nuclear staining (blue). The co-localization of the two proteins is given by the yellow tint, in Leydig cells (asterisk) and spermatogonia (arrowheads). PREP localizes also in the Sertoli cells cytoplasm (arrows). The images in the insets were captured at X40 magnification, all the others at X20 magnification. Scale bars represent 20 μm, except for the insets, where they represent 10 μm.
  • Fig. 2: Immunofluorescence co-localization analysis for GluA2/3 and p-ERK1/2 in rat testis from controls (a) and at 30 min (b), 2 h (c) and 5 h (d) after D-Asp acute treatment. a, b, c, d: GluA2/3 fluorescence (green), p-ERK1/2 staining (red) and DAPI-fluorescent nuclear staining (blue). The co-localization of the two proteins is given by the yellow tint, in Leydig cells (asterisk) and spermatogonia (arrowheads). p-ERK1/2 localize also in the Sertoli cells cytoplasm and in spermatozoa. The images in the insets were captured at X40 magnification, all the others at X20 magnification. Scale bars represent 20 μm, except for the insets, where they represent 10 μm.
  • Fig. 3: Immunofluorescence co-localization analysis for GluA2/3 and p-Akt in rat testis from controls (a) and at 30 min (b), 2 h (c) and 5 h (d) after D-Asp acute treatment. a, b, c, d: GluA2/3 fluorescence (green), p-Akt staining (red) and DAPI-fluorescent nuclear staining (blue). The co-localization of the two proteins is given by the yellow tint, in Leydig cells (asterisk) and spermatogonia (arrowheads) and spermatocytes. p-Akt localize also in the Sertoli cells cytoplasmic protrusions. The images in the insets were captured at X40 magnification, all the others at X20 magnification. Scale bars represent 20 μm, except for the insets, where they represent 10 μm.
  • Fig. 4: Specificity controls of the used antibodies. A: Western blot analysis carried out on total protein extracts from control rat testis, using PREP antiserum alone (left lane) or preadsorbed 5fold excess with the epitope (right lane). B: Immunofuorescence staining in control rat testis. PREP specificity control was checked by preadsorbing primary antiserum with of the corresponding epitope. C: IgG isotype controls, obtained using rabbit IgG serum at the same concentration as antibodies (a-e); negative controls, obtained by omitting the primary antibody (f-j). a,f: controls; b,g: 30 min D-Asp; c,h: 2h D-Asp; d,i: 5h D-Asp; e,j: 15 days D-Asp. The scale bars in B an C represent 20 μm.

 

    Society for Reproduction and Fertility

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    Expression and localization of PREP and GluA2/3 after chronic D-Asp treatment. (A) Western blot analysis of PREP and GluA2/3 expression in the testis from D-Asp-treated and control rats. (B) The amount of PREP was quantified using ImageJ program and normalized with respect to β-actin (see A). (C) The amount of GluA2/3 was quantified using the ImageJ program and normalized with respect to β-actin (see A). Values represent the means ± s.d. of five samples (two bands shown in upper panel). *P < 0.05 vs controls. (D). Localization of PREP and GluA2/3 in the testis from D-Asp-treated rats (b, d) and controls (a, c). a, b: PREP fluorescence (green), DAPI-fluorescent nuclear staining (blue), PNA lectin acrosome staining (red). c, d: GluA2/3 fluorescence (green), DAPI-fluorescent nuclear staining (blue), PNA lectin acrosome staining (red). The images in the insets were captured at ×40 magnification, all the others at ×20 magnification. Scale bars represent 20 μm, except for the insets, where they represent 10 μm. Asterisks: Leydig cells; Arrowheads: Spermatogonia; Arrows: Sertoli cells.

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    Expression of p-ERK1/2 and p-Akt after chronic D-Asp treatment. (A) Detection of P-ERK1 and P-ERK2 proteins in the testis from D-Asp-treated rats and controls by Western blot. (B) The amounts of phosphorylated ERK1 and ERK2 were quantified using ImageJ program and normalized with respect to ERK1 and ERK2, respectively (see A). Values represent the means ± s.d. of five samples (two bands shown in upper panel). *P < 0.05 vs controls. (C) Western blot analysis of Akt protein in the testis from D-Asp-treated and control rats. (D) The amount of phosphorylated Akt was quantified using the ImageJ program and normalized with respect to Akt (see C). Values represent the means ± s.d. of five samples (two bands shown in upper panel). *P < 0.05 vs controls.

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    Expression and localization of PREP after acute D-Asp treatment. (A) Detection of PREP protein by Western blot in the testis of D-Asp-treated and control rats. (B) The amount of PREP was quantified using the ImageJ program and normalized with respect to β-actin (see A). Values represent the means ± s.d. of five samples (one band shown in the upper panel). *P < 0.05 vs controls. (C) Immunofluorescence analysis for PREP in rat testis from controls (a) and at 30 min (b), 2 h (c) and 5 h (d) from D-Asp treatment. a, b, c, d: PREP fluorescence (green), DAPI-fluorescent nuclear staining (blue), PNA lectin acrosome staining (red). The images in the insets were captured at ×40 magnification, all the others at ×20 magnification. Scale bars represent 20 μm, except for the insets, where they represent 10 μm. Asterisks: Leydig cells; Arrows: Sertoli cells.

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    Expression and localization of GluA2/3 after acute D-Asp treatment. (A) Detection of GluA2/3 by Western blot in rat testis at various times after D-Asp treatment. (B) The amount of GluA2/3 was quantified using ImageJ program and normalized with respect to β-actin protein (see A). Values represent the mean ± s.d. of five separate experiments (one band shown in upper panels). *P < 0.05 vs controls. (C) Immunofluorescence analysis for GluA2/3 in rat testis from controls (a, c) and at 5 h after D-Asp treatment (b, d). a, b, c, d: GluA2/3 fluorescence (green), DAPI-fluorescent nuclear staining (blue), PNA lectin acrosome staining (red). The images in the insets were captured at ×40 magnification, all the others at ×20 magnification. Scale bars represent 20 μm, except for the insets, where they represent 10 μm. Asterisks: Leydig cells; Arrowheads: Spermatogonia.

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    Expression of p-ERK1/2 and p-Akt after acute D-Asp treatment. (A) Analysis of rat testes with anti-phospho-ERK1/2 antibodies showing phosphorylation and activation of ERKs at various times after D-Asp treatment. Two specific bands, corresponding to the sizes of 44 kDa and 42 kDa, respectively, were detected. (B) The amounts of activated ERK1/2 were quantified using ImageJ program and normalized with respect to total ERK1/2. Values represent the mean ± s.d. of five separate experiments (one band shown in upper panels). *P < 0.05 vs controls. (C) Western blot analysis of rat testes with anti-phospho-Akt antibody showing phosphorylation and activation of Akt at various times after D-Asp treatment. (D) The amount of activated Akt was quantified using ImageJ program and normalized with respect to total Akt. Values represent the mean ± s.d. of five separate experiments (one band shown in C). *P < 0.05 vs controls.

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